Gp96 heat shock proteins (HSPs) elicit immunity specific to the tumors from which they are isolated, but not to antigenically distinct tumors. Gp96 isolated from normal tissues does not elicit immunity to tumors. No differences in sequence or glycosylation are seen in gp96 between tumors and normal tissues or among tumors. In view of the lack of such differences in spite of antigenic specificity and in view of the observations that HSPs are known to bind peptides, we propose that gp96 is not immunogenic per se, but is a carrier of antigenic peptides. As gp96 resides largely in the endoplasmic reticulum (ER), we further suggested that it acts as peptide-acceptor in the ER and may be accessory to charging of MHC class I molecules. We also observe that: A number of peptides can be eluted from gp96 by acid-stripping. Gp96 contains ATP-binding casettes, binds ATP in vitro and in vivo and is anATPase. Immunization with gp96 elicits specific CTLs to tumors and virus-infected or transformed cells. Addition of gp96 to RMA-S extracts facilitates assembly of MHC class I - beta2 micro-globulin. Similar to other elements of class I presentation, gp96 is up-regulated by gamma-interferon. We propose to continue to examine our hypothesis using the 6138 tumor of C3H mice, where we show that immunization with gp96 derived from 6138 elicits 6138-specific CTLs. We aim to: 1. Characterize 'native' peptides eluted from tumor-derived gp96: - separate peptides and determine their mass and sequence by 2-d mass spectroscopy; 2. Characterize peptide - binding by gp96 in vitro: - test binding of gp96 with peptides known to be recognized by T cells; - test the immunogenicity of 'empty' and reconstituted gp96 molecules; 3. Identify the gp96-eluted antigenic peptide and the protein from which it is derived: - sensitize C3H fibroblasts with 6138-gp96-eluted peptides for lysis by tumor (6138)-specific CTLs; use this assay to characterize the antigenic peptide and its parent protein; - isolate the gene for this protein and characterize the tumor-specific alteration; 4. Define the immunological basis of gp96 - elicited tumor immunity: - deplete gp96 - immunized mice of macrophage, CD4+ or CD8+ cells and monitor consequence on CTL generation and tumor immunity; - reconstruct the circuitry of gp96-elicited immune response by adoptive transfer experiments; These studies will serve as prototypes for identification of antigenic epitopes of other tumors.